Edema is the term generally used to describe the accumulation of excess fluid in the intercellular (interstitial) tissue spaces or body cavities. Edema may occur as a localized phenomenon such as the swelling of a leg when the venous outflow is obstructed; or it may be systemic as in congestive heart faillure or renal failure. When edema is severe and generalized, there is diffuse swelling of all tissues and organs in the body and particularly pronounced areas are given their own individual names. For example, collection of edema in the peritoneal cavity is known as "ascites"; accumulations of fluid in the pleural cavity are termed "hydrothorax"; and edema of the pericardial sac is termed "pericardial effusion" or "hydropericardium". Non-inflammatory edema fluid such as accumulates in heart failure and renal disease is protein poor and referred to as a "transudate". In contrast, inflammatory edema related to increased endothelial permeability is protein rich and is caused by the escape of plasma proteins (principally albumin) and polymorphonuclear leukocytes (hereinafter "PMNs") to form an exudate.
Edema, whether inflammatory or non-inflammatory in nature, is thus an abnormality in the fluid balance within the microcirculation which includes the small arterioles, capillaries, and post-capillary venules of the circulatory system. Normal fluid balance and exchange is critically dependent on the presence of an intact and metabolically active endothelium. Normal endothelium is a thin, squamous epithelium adapted to permit selective, rapid exchange of water and small molecules between plasma and interstitium; but one which limits the passage of plasma proteins with increases in protein size.
The endothelial lining of all arterioles and venules, and most capillaries in the body, is of the continuous type, having an unbroken cytoplasmic layer with closely apposed intercellular junctions. Physiological studies [Renkin, E., Circ. Res. 41:735-743 (1977); Renkin, E., ACTA Physiol. Scand. (Suppl.) 463:81 (1979); Bottaro et al., Microvasc. Res. 32:389-398 (1986)] have demonstrated normal endothelial permeability for water and small molecules by the existance of water-filled small pores approximately 6 nanometers (hereinafter "nm") in radius or by slits about 8 nm wide. There is also believed to be a system of larger sized pores about 25 nm in radius which accounts for the small quantities of protein and other large solutes that normally cross the endothelial wall barrier.
A variety of different disturbances can induce a condition of edema. These include: an elevated venous hydrostatic pressure which may be caused by thrombosis of a vein or any other venous obstruction; hypoproteinemia with reduced plasma oncotic pressure resulting from either inadequate synthesis or increased loss of albumin; increased osmotic pressure of the interstitial fluid due to abnormal accumulation of sodium in the body because renal excretion of sodium cannot keep pace with the intake; failure of the lymphatics to remove fluid and protein adequately from the interstitial space; an increased capillary permeabiity to fluids and proteins as occurs in the inflammatory response to tissue injury; and an increased mucopolysaccharide content within the interstitial spaces.
Currently accepted therapeutic treatments for edema include those biogenic and synthetic pharmacological agents used to treat generalized inflammations, of which edema is merely one clinical manifestation. Such agents are said to inhibit the synthesis of pro-inflammatory (pro-phlogistic) metabolites; and can include such agents as aspirin, ibuprofen (salicylates and propionate derivatives), steroids, and anti-histamines. These agents have a wide scale of effectiveness and, in general, are most valuable in the treatment of minor inflammatory problems that produce only minor, localized edemas. There are few, if any, agents that are therapeutically effective in the treatment of severe, local and systemic edemas. Furthermore, as far as is known, there is no effective agent or admixture in present use as a prophylactic against these conditions.
It will be noted that until recently the endothelial cells, which constitute the microvasculature, were considered to be functionally passive in nature; in-vivo fluid exchange at the level of the microvasculature was therefore also considered to be functionally passive. Only in 1967 was it proposed that the passage of fluid and solute might occur at interendothelial junctions within the microvasculature, a routing also known as the "paracellular" pathway [Karnovsky, M. J., J. Cell Bio. 35:213-236 (1967)]. Subsequent investigations have focused principally upon other aspects of microvascular structural integrity such as: vesicle transport or transcellular channels which regulate the distribution of integral membrane proteins [Singer and Nicolson, Science 175:720-731 (1972)]; the presence of stress fibers intracellularly which are microfilament bundles composed of actin, myosin, and other contractile proteins [Fujiwara and Pollard, J. Cell. Biol. 71:848 (1976)]; the ability to disrupt endothelial actin cytoskeleton using cytochalasin B with a resulting increase in permeability for plasma proteins [Shahby et al., Circ. Res. 51:657-661 (1982)]; the demonstration that serotonin, histamine, and norepinephrine at physiological titers and concentrations inhibit endothelial cell movement [Bottaro et al., Am. J. Physiol. 248:C252-C257 (1985)]; the demonstration that serotonin and norepinephrine stimulate the assembly of stress fibers within endothelial cells whereas histamine produces stress fiber disassembly [Welles et al., J. Cell. Physiol. 123:337-342 (1985) and Inflammation 9:439-450 (1985)]; and, an in-vitro assay which demonstrates that endothelial cells are more effective as a barrier to impede labelled albumin diffusion when compared with cell cultures of vascular smooth muscle or fibroblasts [Bottaro et al., Microvasc. Res. 32:389-398 (1986)]. Such investigations have been directed at elucidating the mechanism of action present within the endothelial cytoskeleton; and identifying the role of the cytoskeleton in maintaining microvascular endothelial motility observable as junctional integrity. All these investigations and publications were therefore concerned with only the formulation of a theoretical model for mechanistic cytoskeleton controls and the accumulation of experimental evidence-to support the existence of such a mechanistic model.
Remote from and completely unrelated to these investigations regarding an active role model for endothelial cells in the microvasculature, were other research efforts directed towards the isolation and identification of the component substances of the poisonous green fungus Amanita phalloides. At least ten peptide-like substances of complex structure have been identified; most of these substances have proven to be extremely toxic liver toxins [Liebig's Ann. Chem., volume 617, page 152, 1958; Pharmacol. Reviews, volume 7, page 87, 1959; Liebig's Ann. Chem., volume 704, page 226, 1967].
Upon isolation and empirical analysis of the naturally occurring individual components of Amanita phalloides, investigators found that one of the naturally occurring substances, antamanide, was not only completely non-toxic of itself, but also was found capable of annulling the toxic effects of fetal doses of phalloidin and/or of protecting the liver completely when administered in therapeutic doses [Wieland et al., Angew. Chem. 80:208 (1968)]. Subsequently, investigations of this cyclic decapeptide, antamanide, have proceeded in two different directions: one research effort involved methods of synthesizing, purifying, and preparing analogues of antamanide. These investigations are exemplified by: U.S. Pat. Nos. 3,705,887 and 3,793,304; Anderson et al., J. Am. Chem. Soc. 88:1338-1339 (1966); Anderson et al., J. Am. Chem. Soc. 89:5012-5017 (1967); Wieland, T., Angew. Chem. (Internat. Edit. ) 7:204-208 (1968); Ovchinnikov et al., Proc. Eur. Pept. Synp. 11:403-415 (1973); Wieland et al., Liebig's Ann. Chem., number 3, pages 371-380, 1977; Burgermeister et al. Eur. J. Biochem. 44:305-310 (1974); Tonelli, A. E., Biochemistry 12:689-692 (1973); Patel, D. J., Biochemistry 12:677-688 (1973); Ivanov et al., Biochem. Biophys. Res. Comm. 42:654-663 (1971); and Bir et al., J. Peptide Protein Res. 13:287-295 (1979)].
In comparison, the other investigative approach focused upon the physiological and pharmacological attributes of antamanide and its synthetic analogues. These investigations are exemplified by the following: Faulstich et al., Hoppe Scylers. Z. Physiol. Chem. 359:1162-1163 (1974); Ovchennikov et al., Experientia 28:399-401 (1972); Wieland et al., Proc. Nat. Acad. Sci. U.S.A. 81:5232-5236 (1984); Carle, I. L., Proc. Nat. Acad. Sci. U.S.A. 82:7155-7159 (1985); Munter, K. D. and H. Faulstich, Biochim. Biophys. Acta 860:91-98 (1986); Nielsen, O, Acta Pharmacol. Toxicol. 59:249-251 (1986); and Raymond et al., Eur. J. Pharmacol. 138:21-27 (1987).
In all of these published investigations and reports, the cyclic decapeptides comprising antamanide (also called "Phallin A") and its analogues were recognized solely as chemical agents capable at very low dosage of counteracting the effects of an absolutely fatal dose of phalloidin or of completely protecting the liver against such a fatal dose of phalloidin. Only recently was there any variation of these investigations into areas concerning cell proliferation and wound healing [Choi et al., FASEB J. 3:A290, Abstract No. 368 (1989)]. This Abstract was the first publication to suggest that antamanide might serve as a therapeutic agent in the treatment of vascular disease. Accordingly, the use of antamanide and the synthetic analogues remains primarily and predominently as an anti-toxin against the effects of phalloidin.